Osteosarcoma is the most common primary bone malignancy of childhood and adolescence, and 40% of patients diagnosed with osteosarcoma die of their disease. Recent studies of leukemia, brain and breast cancer, indicate that a small subpopulation of cells of stem-like cells, is responsible for tumorigenesis and malignancy while the remaining tumor mass is comprised of differentiated progeny of the tumor stem cell that are relatively benign. We have initiated studies to determine the participation of stem-like cells in the tumorigenesis of osteosarcoma. In preliminary investigations we found evidence of stem-like cells by three criteria: (i) Sphere Formation: A subset of osteosarcoma cells form spherical colonies and self-renew, (ii) Embryonic Stem Cell (ESC) Markers: "sarcospheres" express marker genes of human ESCs, such as Oct-4, Nanog and SSEA4. (iii) Mesenchymal Stem Cell (MSC) Markers: Cells in osteosarcomas bear cell surface markers, Stro-1 and other antigens typically associated with MSCs and bone marrow stromal cells and can be induced to differentiate along at least two mesenchymal lineages. [unreadable] For the present study we will test the over-arching hypothesis that stem-like cells in osteosarcoma are tumorigenic in vivo. We will work to address this hypothesis through two specific aims that pursue distinct yet complementary experimental approaches: [unreadable] Specific Aim 1: will test the hypothesis that the sphere culture system preferentially selects for tumorigenic cells in osteosarcomas. Although the sphere culture system has enabled the identification of cells with the ability to form spheres and self-renew, we need to establish a direct link between the ability a cell to form spheres and its ability to form tumors. For this, cells from several sources of osteosarcoma will be isolated and cultured in the sphere system and in monolayer. Cells from the respective cultures will be xenografted into immunocompromised (NOD/SCID) mice and the relative tumor forming capacity of sphere vs. monolayer cells will be evaluated for each osteosarcoma. [unreadable] Specific Aim 2: will test the hypothesis that cells in osteosarcomas that possess attributes of ESCs or MSCs are tumorigenic. Using Oct-4 and Stro-1 expression as indicators of ESC- and MSC-like cells, respectively, we will compare in the NOD/SCID mouse model the relative tumor forming capacity of osteosarcoma cells enriched and depleted for these individual markers. [unreadable] The results of our experimental plan should provide a clear demonstration of the contribution of stem-like cells in the tumorigenesis of osteosarcomas and provide a foundation upon which to base large scale investigations suitable for an R01 level application [unreadable] [unreadable] [unreadable]